Hepatitis C virus (HCV) infects more than 170 million people worldwide and is the leading condition requiring liver transplantation. There is no vaccine and current treatments are often ineffective and have severe side-effects. There is currently considerable interest in the use of RNA interference as an antiviral modality. Although both siRNAs and shRNA can be highly potent, with IC50s in the subnanomolar range, the ability to deliver effective amounts of an RNA drug to infected hepatocytes in the liver limits efficacy. Hence, absent a breakthrough in delivery technology, si/shRNAs with still higher potency are needed if they are to be clinically useful. In addition, while delivery to the liver by an intravenous route is easier than for many other tissues, every delivery mode allows some uptake by other organs, leading to potential adverse effects in uninfected tissues. Here we propose a novel approach in which limited intracellular delivery is compensated by a mechanism for production of many shRNAs from each therapeutic molecule. Moreover, the production of shRNAs occurs only in cells infected by HCV. Hence the therapeutic molecule can be designed to target either a viral or a host gene, resulting in either suppression of viral growth or death of the infected cell (or both in a combination therapy). No functional drug is formed when the agent is taken up by uninfected cells, so adverse effects from a treatment using this approach should be minimal. Thus, the method provides two advantages over direct delivery of siRNAs or shRNAs: reduced toxicity as well as increased potency through an amplification mechanism. The strategy, which does not require gene therapy, can potentially be extended to targeting most if not all RNA viruses. Project Narrative: Hepatitis C virus (HCV) infects some 2-4% of the US population and more than 170 million people worldwide and can lead to cirrhosis or liver cancer. There is no vaccine and current treatments are often ineffective and have severe side-effects. While potential new treatments using RNA interference have generated some excitement, they are currently limited in potency. The proposed study will investigate a novel therapeutic approach to HCV and many other RNA viruses that has the potential to overcome the potency limitation by using a mechanism for amplifying the effects of the drug specifically in infected cells.